My name is Ed Fischer, and I'm a retired professor of biochemistry
at the University of Washington. Today I would like to give you
a sort of rapid historical overview of the discovery of reversible
protein phosphorylation as a regulatory mechanism.
Now this came about in the course of a study that
we had undertaken with my colleague and longtime friend,
Ed Krebs, on glycogen phosphorylase 60 years ago. The
original reaction we described was really embarrassingly
simple. And nobody would have paid much attention to it
if it didn't turn out to be absolutely crucial for the regulation
of cellular processes. On the other hand, it's fair to say that when we
came up with it, it came as a total surprise because in those days
one knew essentially nothing about cell and enzyme regulation.
The prevailing idea was that enzymes were regulated
by the rate at which they were synthesized and then degraded.
And that stood until the late '40s and early '50s, when it was
realized that this couldn't be the case. That cells had to have
ways of manipulating the activity of their enzymes once they
had been produced and liberated inside the cell. And that
was really one of the big problems that faced the biologists in those
days and that was the problem that we decided to tackle
with Ed. We wanted to know how the activity of an important
enzyme called glycogen phosphorylase was regulated.
It was very important because it was already known to catalyze
the rate limiting step of carbohydrate metabolism. The first step in the
degradation of glycogen. And furthermore, it was under complex
hormonal regulation, being activated by adrenaline and
inhibited by insulin. When first discovered in the early '30s,
by Yakov Parnas in Poland and Carl and Gerty Cori in the United States,
the muscle enzyme was known to be totally inactive in the absence
of AMP, adenylic acid. And automatically, the Coris assumed that
AMP served as a coenzyme, and that stood until about '43,
when Arda Green and Coris crystallized the native form
of the active form of the enzyme. But she crystallized it in a new form.
In that it was fully active in the absence of AMP. So they called that
form phosphorylase a and logically assumed that it had to contain
AMP bound covalently to the protein as a prosthetic group.
And furthermore, they assumed that that had to be the native
form of the enzyme in muscle, because when muscle extracts
were left standing, it was rapidly converted to the old form
that they now call phosphorylase b, the form that required AMP
for activity. But they could show no formation of AMP in the reaction
and furthermore, they could show no AMP in the native
enzyme. So they were stuck. The Coris knew that phosphorylase
existed in two forms, active and inactive, but they did not
know in which way those two forms differed. And as strange as it would
seem today, they actually dropped that problem. Ed and I became
involved in that problem when we found ourselves in the same department
in Seattle. Ed had been a postdoc of the Coris and had some
hesitations to work on that problem. In those days, it should be said
that students and postdocs when they left the lab did not
work on the problem of their mentors. But then he reasoned that he had left
St. Louis five years ago and it should be permissible for us to work
together on that problem. Okay, so we cleared the bench in the lab and started
working, just the two of us side by side, much more like two friends than
like two colleagues in the department. And the first thing we had to do
of course was to purify phosphorylase, get the pure enzyme,
by Coris' classical procedure. Now Coris' procedure consisted in
taking rabbit muscle, grinding it up, extracting the hamburger in
water, throwing the whole thing in cheesecloth, squeezing
the cheese cloth, and then clarifying the very turbid
extract by passing it through a battery of filter papers.
And of course the filter paper became clogged up right away,
so with a glass rod, they would punch a hole and go to the next
paper, and then the next one. It wasn't unusual for them to use
maybe 15-20 filter papers for one purification. The method
was totally archaic and in fact, here's a picture of our lab.
So, we decided with Ed, enough with that jazz. Let's replace
paper filtration by a centrifugation. In defense of the Coris,
it should be said that in those days there was no good
lab centrifuge. But then, when we centrifuged our extract,
we could never obtain active phosphorylase a, only the
so-called "degraded b form." Until finally, in desperation,
we said, you know we have to go back to Coris' procedure.
Following it to the letter, paper filtration, and all. Measuring
every step to see what's going on. And to our immense
surprise, this is what we found. That the very first extract from
the muscle, which should have contained phosphorylase a,
contained the inactive form of the enzyme. And then when it passed
through the filter paper step, it was converted to active
phosphorylase a. Speaking of a let down would be to speak
it easily, because paper filtration as a method to activate an enzyme
was really the pits. Pretty below the worst. But, it soon happened
that it was not the filtration perse that did the trick, but
the fact that all filter papers in those days were contaminated
by calcium ions. And enough was picked up during those multiple
filtrations to convert inactive to active phosphorylase. And then
quite rapidly, we found that ATP was always present in crude muscle
extract was also needed. And that strongly suggested that we
were dealing with a phosphorylation reaction. Of course we didn't know what
had become phosphorylated, because in those days you had
no ATP 32, you had to synthesize the damn thing yourself.
But we knew that Art Kornberg in St. Louis had made some for his
beautiful studies of DNA synthesis. So we called Art and he
right away sent us a sample of gamma-labeled ATP 32,
with which we could show that radioactivity was incorporated
in a protein fraction, which we could isolate and which turned out
to be phosphorylase. So then for the first time, we could then
suggest that the activation of phosphorylase occurred like so.
That the inactive enzyme was converted, was phosphorylated,
converted to the active form in a reaction that required calcium,
magnesium, and ATP, and an enzyme of course, which we called
phosphorylase kinase. And then the reverse reaction happened to be
catalyzed by a phosphorylase phosphatase. And immediately
we shipped the paper to the Journal of Biological Chemistry.
And we knew that Carl Cori was one of the editors, so you know,
perhaps a question, but then we had faith that Cori would not
take advantage of that situation. And he did not, in fact he was very
excited by our results and he never touched that problem.
Now, the reaction that I showed earlier became slightly more complicated
when we began to use partially purified elements. Particularly
when we used partially purified phosphorylase kinase.
Because it became clear that calcium could not act in
that reaction. And yet, it was absolutely needed when crude
muscle extracts were used. So the only possibility is
that in crude muscle extract, calcium had to act at an earlier
step. And we hypothesized that perhaps phosphorylase kinase
just like phosphorylase, might also exist in an inactive and
active form. And perhaps calcium was required for that reaction,
and that turned out to be correct. In the presence of calcium
ions, phosph kinase is rapidly phosphorylated and activated.
At that same time, Earl Sutherland, Ted Rall, and his group
had isolated cyclic AMP, which they showed had served as
a second messenger, intracellular messenger, for the action of
adrenaline. So Earl immediately sent us a sample of his
material, and we could show that it acted on the activation
of phosph kinase. Either by accelerating the rate of autophosphorylation
or perhaps even, by acting on yet another enzyme, which for want of a better
term, we called a kinase kinase. Some people thought it was a funny type
of name, others thought it was a dumb name. And we never forget the
comments of the reviewer who looked at this paper, first of all, he
didn't believe that another enzyme would be involved. He said,
when will it end? But then we said, if really another enzyme
is involved, I wish that the authors would think of a better name than
a kinase kinase. Anyway, that hypothesis was confirmed 4 years
later by the isolation, by Ed and Don Walsh, of the cyclic AMP
dependent protein kinase. And since by that time, Earl and his
group had clarified the production of cyclic AMP at the membrane,
the whole cascade for the phosphorolysis of glycogen was
elucidated. We have been often asked by Ed whether we realized
at the beginning that we were dealing with a very ubiquitous
and therefore very fundamental reaction. Absolutely not.
We knew that it was a nice reaction, we knew that it was an
important and exciting one, we stayed with it, but we could
never have guessed the incredible developments that followed.
Should be said that 4-5 years later, everybody was thinking in terms of
allostery. Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux
had come up with a model of allosteric activation of enzymes.
And it was very clear to me that Jacque Monod was a very
close friend of mine, never believed one second that covalent
modification by protein phosphorylation could serve a fundamental
role in the regulation of cellular processes. We know today
that phosphorylation is one of the most prevalent mechanisms of regulation,
and it is clear that it would be very difficult to find a physiological
reaction that was not directly or indirectly affected by protein
phosphorylation. Another question that was often asked
was how was it for you guys to work for more than 50 years
together? Well, it was very easy and most enjoyable.
At first, Ed would make fun, would laugh at my English. Every morning,
I would say, "let's go take a coffee." Because in French we say,
prendre un cafe. And he really made fun of that.
What Ed didn't realize was that in the following year,
while my English didn't improve very much, his deteriorated
completely. Anyway, those were marvelous, marvelous days.
And during all the time we worked together, we never considered
what we were doing as work. It was never work.
It was fun. Thank you.